Project Summary Bacterial infection is sensed by evolutionarily conserved pattern recognition receptors, including Toll Like Receptors (TLRs). TLR signaling induces production of inflammatory mediators that play a key role in controlling infection as well as in the pathologic sequellae of infection, including gram-negative sepsis. Recent studies in our laboratory and others revealed a role for the cysteine protease, caspase-8 (Casp8) as an important regulator of the transcriptional response to bacterial infection. Casp8-deficient mice and humans are highly susceptible to mucosal bacterial infection, implicating Casp8 as a key regulator of anti-bacterial immune defense. Intriguingly, our preliminary studies demonstrate that Casp8 plays a cell-intrinsic role in regulating transcription of key anti-microbial inflammatory mediators in response to gram-negative bacterial infection and TLR stimulation, and this is entirely independent of the well-established role of Casp8 in regulating cell death. Autoprocessing of Casp8 at a key aspartate residue leads to stabilization of the enzymatically active Casp8 homodimer, and subsequent cleavage of its apoptotic substrates. Conversely, Casp8 functions as a heterodimer with cFLIP to limit cell death and promote cell survival. We have now generated a novel Casp8DA mutant mouse in which the ability of Casp8 to homodimerize is eliminated, and have found that Casp8DA macrophages have a significant defect in their ability to induce inflammatory cytokine production in response to TLR engagement. These studies provoke the conceptually novel hypothesis that the Casp8 homodimer plays a non-apoptotic role in the induction of antimicrobial responses during bacterial infection or TLR engagement. We propose two Specific Aims to address this important gap in our knowledge. First we will utilize newly- generated mutant mice that distinguish between the function of the caspase-8 homo- and hetero-dimers to define the define molecular mechanism of caspase-8-mediated control of inflammatory cytokine gene expression. In particular we will test whether caspase-8 regulates inflammatory gene expression by cleaving and inactivating transcriptional repressors or activating a transcriptional inducer. Second, we will use well- defined murine models of systemic bacterial infection to interrogate the in vivo role of caspase-8-dependent inflammatory cytokine production in innate and adaptive antimicrobial immune defense.